title: A safe and efficient method to retrieve mesenchymal stem cells from three-dimensional fibrin gels.
abstract: Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro and eventually cease to retain any resemblance to in vivo resident MSCs. Three-dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain the cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate, in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has a strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable but also retain their proliferative and multilineage potentials. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.
authors.affiliation: 1 Department of Biomedical Engineering, University of Michigan , Ann Arbor, Michigan.
authors.forename: Bita
authors.initials: B
authors.lastname: Carrion
authors.forename: Isaac A
authors.initials: IA
authors.lastname: Janson
authors.forename: Yen P
authors.initials: YP
authors.lastname: Kong
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Putnam
mesh_terms: D050156:Adipogenesis / Q000187:drug effects / Q000235:genetics; D019070:Cell Lineage / Q000187:drug effects / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D005337:Fibrin / Q000494:pharmacology*; D005782:Gels / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D016133:Polymerase Chain Reaction; D013381:Subtilisins / Q000494:pharmacology
chemical_list: D005782:Gels; D005337:Fibrin; D013381:Subtilisins; C053771:nattokinase